The Role of Surveillance Inspections in Reducing False-Positives of SARS-CoV-2 Omicron Variants during the COVID-19 Epidemic

Objective This study aims to assess the effectiveness of surveillance inspections conducted by the provincial health committee in Quanzhou city during a COVID-19 outbreak in reducing false-positive results in SARS-CoV-2 RT-PCR assays. Method The team conducted on-site inspections of laboratories that participated in mass screening, recording any violations of rules. Results The positive cases in five rounds of mass screening were 23, 173, and 4 in Licheng District, Fengze District, and Luojang District, respectively. The false-positive rates in the five rounds of mass screening were 0.0099%, 0.0063%, 0.0018%, 0.0006%, and 0%, respectively. The study also recorded that the number of violations in the seven selected laboratories was 36, 68, 69, 42, 60, 54 and 47. The corresponding false-positive rates were 0.0012%, 0.0060%, 0.0082%, 0.0032%, 0.0060%, 0.0027%, and 0.0021%, respectively. The study found a positive correlation between false-positive rates and the number of violations (r = 0.905, P=0.005), and an inverse correlation between false-positive rates and the frequency of surveillance inspections (r = −0.950, P < 0.001). Conclusion Daily surveillance inspection in laboratories can remind laboratories to strictly comply with standard procedures, focus on laboratory quality control, and reduce the occurrence of false-positive cases in SARS-CoV-2 nucleic acid tests to some extent. This study recommends that government decision-making departments establish policies and arrange experts to conduct daily surveillance inspections to improve laboratory quality control.


Introduction
COVID-19 is still in pandemic status and a public health emergency of international concern. Te omicron variant is believed to have shorter serial interval (2.2-3.5 days) [1][2][3] and a shorter incubation period (2.9-3 days) than the delta variant [2,4,5]. Moreover, it has a higher secondary attack rate [6,7] and has been proven to evade immune surveillance [4,[8][9][10]. Although omicron variant infection may be less likely to cause severe diseases and fatality, this specifc variant can easily spread between individuals [11]. Te impact of the omicron variant should not be ignored since it has a higher transmissibility, and if it spreads within a large population, a greater number of people could develop severe diseases. Additionally, some foreign countries have been observed in hospitalization rate and mortality related to the omicron variant [12]. Over the past two years, China has consistently implemented the dynamic zero-SARS-CoV-2 policy to prevent new outbreaks. However, in recent days, the domestic epidemic has signifcantly intensifed, presenting complicated challenges to this policy. Laboratory tests for SARS-CoV-2 play a critical role in identifying the source of transmission and assisting government countermeasures. To further improve the quality of nucleic acid tests and identify potential issues, the Joint Prevention and Control Organization of the State Council has dispatched surveillance inspection teams to provinces experiencing outbreaks, to conduct problem-oriented surveillance inspections.
On March 13, 2022, nine people tested positive for COVID-19 during routine screening of key groups of people in Fengze District, Quanzhou City. All of the positive cases were hotel staf working for the same hotel. Genome sequencing of the positive samples revealed a sublineage that had evolved from the Omicron BA.2 mutant, and no highly homogeneous sequences were found when compared to other known sequences identifed in China. In response, the provincial health committee dispatched a surveillance inspection team to high-risk areas of Quanzhou City. Te team conducted surveillance inspections on compliance with operation manuals, personal protection, quality control, and cross contamination. Te aim of these inspections was to improve quality control and eliminate the possibility of issuing inaccurate reports. On the fourth day after the deployment of the surveillance inspection team, 105 sample tubes tested positive, and on the ffth day, 159 sample tubes tested positive. Te question remains whether there were any false-positive events in the reports. A review of 37 external quality assessments found that the false-positive rate in detecting RNA viruses ranged from 0.6% to 8.1% [13]. A false-positive result can have a negative impact on a patient's mental and physical health, and misdiagnosis can lead to mismanagement of medical resources. To the best of our knowledge, there has been no report on the evaluation of surveillance inspections on SARS-CoV-2 nucleic acid tests. Tis study aims to assess whether surveillance inspections can reduce false-positive events. March 13, 2022, to April 6, 2022, PCR was used to perform the SARS-CoV-2 nucleic acid test on individual samples from 6.676 million subjects. A false-positive result was defned as a sample that tested positive in the frst test but was found to be negative upon immediate retesting of the same fresh sample and testing in another laboratory. A total of 1.345 million people were screened in Licheng District, Fengze District, and Luojang District. Te remaining 5.331 million people in Shishi City, Jinjiang City, Nanan City, Huian County, Quanzhou Economic and Technological Development Area, and Taishang Investment Area were also tested. In total, the entire city underwent fve rounds of mass screening for COVID-19 between March 20, 2022, and March 28, 2022. During each round of mass screening, seven laboratories located in Licheng District, Fengze District, and Luojang District tested an average of 1,663,000 samples. Given the sample size and the geographic importance of these laboratories, the provincial surveillance inspection team conducted several rounds of inspections.

Inspection Approaches.
Te national molecular laboratory adopted the national standard "Medical institutions Novel Coronavirus nucleic acid Detection Manual" which is the national standard for carrying out novel coronavirus nucleic acid detection, and Quanzhou followed suit. Te inspection list of supervising and testing experts was also based on this national standard. All nine supervisors from the province have held senior titles and have been engaged in molecular diagnosis for more than 10 years. Te surveillance inspection team consisted of nine specialists who adopted a "circuit and on-site rotation" approach to conduct surveillance inspections in 26 laboratories. Tese laboratories had a total workload of 150 rounds of mass screening (an average of six rounds of tests for each laboratory) within 27 days.
Te nine specialists employed a unifed "Key Points for Quality Control Inspectors of SARS-CoV-2 Nucleic Acid Tests" as the inspection tool, and they recorded any violations of rules and sent to the provincial health committee. Laboratories were inspected on a daily basis. Information on positive and false-positive results during the fve rounds of mass screening from March 20 to 28 was collected from seven laboratories out of 26 laboratories. Laboratory one, laboratory three, and laboratory four are located in Licheng District, laboratory two and laboratory fve are located in Fengze District, and laboratory six and laboratory seven are located in Luojang District.

Positive Criteria and False-Positive Criteria.
To be confrmed as positive cases, laboratory results must meet one of the two conditions. Te frst condition is that the real-time fuorescent RT-PCR test results of both ORF1 and N target genes of SARS-CoV-2 in the same sample are positive. If only one target gene tests positive, the sample must be retested or resampled for review. Te second condition is that if the real-time fuorescence RT-PCR test results of a single target gene are positive in two samples of the same type, or if the test results of a single target gene are positive in two diferent types of samples taken at the same time, then the sample can be considered positive. For a positive sample, one to two more sensitive and amplifed nucleic acid test reagents in diferent regions are used to retest the original sample.
If a positive sample from the same laboratory is retested by the Chinese Center for Disease Control and Prevention (CDC) and the result is positive, it is considered a true positive. If the retest is negative or the resampling and reexamination is negative, the sample is considered a false positive according to local policy. When a positive sample from the same laboratory is negative after CDC resampling, it is also considered a false positive. Samples with nucleic acid detection Ct value ≤ 32 undergo whole-genome sequencing by the CDC.

Statistical Analysis.
Statistical analyses were conducted using SPSS version 20.0 (SPSS, Chicago, IL, USA). Te normal distribution of continuous variables was examined using the Shapiro-Wilk test. Te Ct values of the ORF1 and N genes conformed to a normal distribution, and a t-test was employed to compare the groups. Te chi-squared test was conducted to identify signifcant diferences in qualitative variables across the groups. Spearman's rank correlation was used to analyze the correlations between the number of violations and the incidence of false-positive results, as well as the number of surveillance inspections and the incidence of false-positive results. A two-sided P value < 0.05 was considered statistically signifcant.

Demographic Characteristics.
According to the information from the local statistical network, the number of individuals under 18 years old in high-risk areas, specifcally Licheng District, Fengze District, and Luojang District, was 95,289, 183,158, and 61,118, respectively. Te number of individuals above 18 years old in these areas was 32,711, 517,842, and 18,688, respectively. With regards to sex distribution, the male populations were 208,067, 336,136, and 128,499, respectively, while the female populations were 219,933, 364,864, and 119,501, respectively.
During the fve rounds of mass screening between March 20 and March 28, there were 23, 173 and 4 COVID-19 patients in Licheng District, Fengze District, and Luojang District, respectively (see Table 1).
Te false-positive rate of SARS-CoV-2 nucleic acid detection in seven laboratories decreased as the duration of the supervisions was extended. Laboratory one, two, and fve had false-positive results for the SARS-CoV-2 nucleic acid test during the initial inspection, and the false-positive rate on March 24 was lower than that on March 22. On March 20, Laboratory three had the highest false-positive rate among the seven laboratories, and the false-positive rates for the four subsequent supervisions gradually declined. Only one false-positive test for SARS-CoV-2 nucleic acid was detected in Laboratory four, six, and seven, with false-positive rates of 0.0016% ± 0.007% on March 22, 0.013% ± 0.009% on March 24, and 0.010% ± 0.010% on March 20, respectively.

Analysis of the Violations of Rules in the Seven
Laboratories. Misconducts that could lead to inaccurate laboratory testing were observed during the fve rounds of surveillance inspections conducted between March 20 and March 28. Te rules violated by the seven laboratories are presented in Table 3. As seen in the table, the most frequently violated rules were A-2, B-3, B-4, D-4, E-1, and F, which were all violated by four of the laboratories. in the inspected laboratories were 0.0012% ± 0.0007%, 0.0060% ± 0.0021%, 0.0082% ± 0.0026%, 0.0032% ± 0.0014%, 0.0060% ± 0.0034%, 0.0027% ± 0.0019%, and 0.0021% ± 0.0021%, respectively. Tere was a positive correlation between the false-positive rate of SARS-CoV-2 Omicron variants and rule violations (r � 0.905, P � 0.005, Figure 2).

Canadian Journal of Infectious Diseases and Medical Microbiology
Spearman's rank correlation was used to analyze the correlation between the false-positive rate and violation of rules. Te false-positive rate of SARS-CoV-2 Omicron variants and violation of rules were positively correlated (r � 0.905, P � 0.005).

Te Efect of Inspections on False-Positive Results.
In the frst round of surveillance inspection, the false-positive rate of SARS-CoV-2 Omicron variants was 0.0099% ± 0.0024%; the rate decreased to 0.0063% ± 0.0019% in the second round, and continued to decrease as the inspections continued. Te rate was 0.0018% ± 0.0011% in the third round, 0.0006% ± 0.0006% in the fourth round, and fnally reached 0 in the ffth round. Te false-positive rate of SARS-CoV-2 Omicron variants and the number of inspections were inversely correlated (r � −0.950, P < 0.001, Figure 3).
Spearman rank correlation was used to analyze the correlation between the false-positive rate and supervision and inspection times. Te false-positive rate of SARS-CoV-2 Omicron variants and the number of inspections were inversely correlated (r � −0.950, P < 0.001).

Discussion
On March 13, a surveillance inspection team was dispatched to the high-risk area and noticed an increase in positive COVID-19 cases. Te nucleic acid test is the primary method used for diagnosing COVID-19, and it provides direct evidence [14,15]. However, if a laboratory operates at or above its full capacity, it may still function at the same quality as usual, or false-positive results may occur. During the surveillance inspection from March 20 to March 28, almost every laboratory involved in the mass screening produced falsepositive results. Te positive rates in the three high-risk areas were highest in Fengze District, followed by Licheng District and Luoyang District. Laboratory three had the highest number of false-positive samples as well as the highest falsepositive rate. Te main reasons behind these high falsepositive events were manual operational errors. Laboratory three had two operating sections: a P2 biosafety laboratory in a fxed location operated by professional medical laboratory technicians and a mobile cabin laboratory operated by scientifc researchers and backups from other health institutions. Terefore, inadequate training may have led to quality issues. False-positive results, in general, were primarily attributed to mislabelling and contamination [16]. Mislabelling and contamination were the primary causes of false-positive results, such as contamination caused by primers or probes in a commercialized PCR kit for SARS-CoV-2 [17].
During our surveillance inspection, we identifed several factors that contributed to false-positive results. Te most common factor was contamination between samples or with a positive control. Inadequate sterilization of sample tubes or failure to sanitize hands or replace gloves after handling a positive control can lead to cross-contamination. When using small pipette tip, inserting the pipette too deeply into the sample tube can contaminate the pipette and the sample. Vigorous vortexing of the sample without sufcient standing time can cause cross-contamination through aerosols. Tese operational details are consistent with fndings reported in the literature [18]. A second common source of false-positive results is amplifcation products. Montgomery et al. also found that the cDNA of SARS-CoV-2 can cause environmental contamination [19]. Contamination can occur if microplates are not sealed after amplifcation or if the seal bag is not sterilized; steam sterilization of the PCR product can then cause product contamination. Failure to follow the rule of moving only in a single direction can result in contamination in multiple areas. Ultimately, the analysis of results can also contribute to false-positive results. Te default threshold set by the manufacturer's software may cause false-positives if there are one or more wells with large fuctuations in the baseline; even a bubble can cause a falsepositive result.
If there are more than a certain number of positive results in a laboratory, the internal quality control should be analyzed, and the population should also be examined. If the population is the same as that previously tested and the amplifcation curve is highly similar, the possibility of contamination should be considered. To prevent the occurrence of false-positive results, the frst priority is to perform testing in accordance with the standard operation procedures. Second, surveillance of contamination should be conducted. Specifcally, four spots in a biosafety cabinet, four random spots in a sample rack, extraction instruments, capremoval instruments, the body of a pipette where the hand grips, and the door handle of a freezer should be regularly tested to prevent cross-contamination between samples. To detect aerosol contamination, sample tubes with RNAseand DNAse-free water should be placed, with the lid open, on benches in the reagent and sample preparation areas, inside biosafety cabinets and extraction instruments, and on the benches with the amplifcation instruments. Te sample tubes with water should be left open for 6 to 8 hours during sample testing and no less than 16 hours during idle time. Subsequently, the sample tubes with water should be vortexed and tested. If sample contamination or aerosol contamination is detected, testing should be suspended, and the laboratory should be completely ventilated and cleaned. Te     [20]. Trough our surveillance inspection, we found that the more laboratories violated the rules, the higher the falsepositive rate was. Te supervising experts communicated with the laboratory staf on-site and provided instruction and demonstrated correct operation. With the increases in the numbers of supervisions and inspections, the incidence of false-positives in the laboratory gradually decreased.

Conclusion
Continued surveillance inspections can improve the quality control of SARS-CoV-2 RT-PCR assays in laboratories, uncover details of irregularities in laboratory operations, and help identify prevention measures targeting laboratory contamination. Tis study analyzed the possible reasons for false-positive results in the preanalytical, analytical, and postanalytical phases of nucleic acid tests. Te insights provided may shed light on operators of SARS-CoV-2 RT-PCR tests reduce the incidence of these false-positive results, as well as unnecessary quarantine and contact tracing. Tis study advocates that government decisionmaking departments formulate policies and arrange for experts to carry out daily surveillance inspections, which is an efective way to improve laboratory quality control.

Data Availability
Data used to support the fndings of this study are available from the author upon request due to ethical issues.

Ethical Approval
Tis study was conducted in accordance with the Helsinki Declaration, and approval for the study was not required in accordance with local/national legislation.

Consent
Informed consent was obtained from all subjects involved in the study.

Conflicts of Interest
Te authors declare no conficts of interest.

Authors' Contributions
Li-Li Liu and Fa-Lin Chen designed the study. Yu-Hong Zheng, You-Quan Zhang, and Shi-Hua Gao collected all the data. Jin-Piao Lin processed the statistical data. Li-Li Liu created the illustrations. Zhi-Lin Luo and Liu-Min Yu helped analyze the data. Fa-Lin Chen revised the article and approved the version submitted. All the authors contributed to this paper. Li Li Liu and Yu Hong Zheng contributed equally to this study.